Method for simultaneously enriching rare ginsenosides by co-fermentation of eutypa lata and panax ginseng and fermentation product

Abstract

The application provides a method for simultaneously enriching rare ginsenosides by co-fermentation of Eurotium cristatum and Panax ginseng and a fermentation product, wherein the Eurotium cristatum is inoculated into a fermentation substrate containing the root of Panax ginseng, and solid fermentation is carried out; the fermentation product contains ginsenosides Rg2, Rg3 and Rh2; the contents of the three rare ginsenosides Rg2, Rg3 and Rh2 in the fermented Panax ginseng raw material are simultaneously improved; the content of Rg2 ginsenoside is increased to about 1.7 times of the original content; the content of Rg3 ginsenoside is increased to about 1.7 times of the original content; and a new ginsenoside Rh2 is obtained, so that the nutritional value and medicinal efficacy of Panax ginseng are increased.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and more specifically, to *Aspergillus cristatus* (…). Eurotium cristatum A method for simultaneously enriching rare saponins by co-fermenting ginseng and ginseng (commonly known as golden flower fungus) and fermentation products. Background Technology

[0002] Ginseng ( Panax ginseng Ginsenosides (CA Meyer) are a traditional and precious Chinese medicinal herb, and their active ingredient, ginsenosides, possess a wide range of pharmacological activities. Among them, rare ginsenosides Rg2, Rg3, and Rh2 have attracted much attention due to their high bioavailability and significant efficacy: Rg3 has been used in anti-tumor adjuvant preparations (trade name: Shenyi Capsules); Rh2 has shown strong tumor cell inhibitory activity in in vitro experiments; and Rg2 has research value in the fields of neuroprotection and anti-fatigue. However, the content of these saponins in natural ginseng is low (usually less than 0.01%), which restricts their in-depth development and application. Therefore, increasing their content through biotransformation technology has become an important research direction.

[0003] Currently, the main methods for preparing rare ginsenosides include chemical hydrolysis, enzymatic conversion, and microbial fermentation. Chemical hydrolysis requires strong acid or high temperature conditions, resulting in numerous side reactions and environmental pollution. Enzymatic conversion offers mild conditions and good selectivity, but a single enzyme cannot cover multiple conversion pathways, and cost and stability limit its large-scale application. Microbial fermentation utilizes complex enzyme systems secreted by microorganisms to achieve saponin structure modification under mild conditions, possessing green, economic, and industrialization potential, and is currently a research hotspot.

[0004] *Aspergillus cristatus*, a fungus used in both food and medicine, is highly safe and has been used in research on ginsenoside transformation. Chinese patent CN202310932714.5 discloses a method for fermenting ginseng with *Aspergillus cristatus*. Experimental data show that the content of ginsenoside Rg3 significantly increased after fermentation (up to 440 times the original content), and Rg2 was listed as a detection indicator, but specific data on its content change were not provided; the method also did not involve the generation and detection of Rh2. Li Sulin et al. (Food Science, 2020) systematically studied the transformation characteristics of ginsenosides by *Aspergillus cristatus*, pointing out that the glycosidase secreted by this strain has strong hydrolytic activity against β-(1-6) glycosidic bonds but weak activity against β-(1-2) glycosidic bonds. Therefore, "Rh2 was not detected" in the fermentation product, and it was stated that "the glycosidase produced by the strain has considerable specificity." This conclusion provides experimental evidence for understanding the transformation characteristics of *Aspergillus cristatus*. However, no literature has clearly reported the simultaneous enhancement of Rg2, Rg3 and Rh2 through a single microbial fermentation system.

[0005] In summary, existing technologies have made positive progress in the field of *Aspergillus cristatus* fermentation of ginseng (such as the efficient enrichment of Rg3), but there is still room for research on the synergistic transformation of multiple rare saponins. Developing a fermentation strategy that can further expand the saponin transformation spectrum of *Aspergillus cristatus* and achieve the synergistic enrichment of Rg2, Rg3, and Rh2 while retaining the safety and green advantages of *Aspergillus cristatus* is of positive significance for improving the high-value utilization of ginseng resources. Summary of the Invention

[0006] The purpose of this invention is to simultaneously increase the content of three rare saponins Rg2, Rg3 and Rh2 in fermented ginseng raw materials by simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A method for simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng, the method comprising:

[0009] *Aspergillus cristatus* was inoculated into a fermentation substrate containing ginseng roots for solid-state fermentation. The fermentation products contained ginsenosides Rg2, Rg3, and Rh2. The content of rare saponins Rg2, Rg3, and Rh2 in the ginseng raw material co-fermented with *Aspergillus cristatus* was higher than that in the unfermented material.

[0010] As a preferred technical solution of the present invention, the fermentation substrate is prepared by mixing fresh ginseng root slices with deionized water and then sterilizing.

[0011] As a preferred technical solution of the present invention, the sterilization conditions are a temperature of 110-120℃ and a time of 10-20min.

[0012] As a preferred technical solution of the present invention, the sterilization conditions are a temperature of 115°C and a time of 15 minutes.

[0013] As a preferred technical solution of the present invention, the *Eurotium cristatum* is inoculated in the form of a spore suspension, and the inoculation amount of the spore suspension is 1-2% of the weight of the fermentation substrate.

[0014] As a preferred technical solution of the present invention, the spore suspension is prepared by the following steps:

[0015] The *Eurotium cristatum* strain was inoculated into potato dextrose agar medium and cultured at 25-30°C for 4-6 days. The spores were scraped off and suspended in sterile water, then shaken to mix.

[0016] As a preferred technical solution of the present invention, the solid fermentation temperature is 20-30℃ and the fermentation time is 7-10 days, until a continuous mycelial layer is formed on the surface of the ginseng substrate.

[0017] As a preferred technical solution of the present invention, the method further includes a step of post-processing the fermentation product:

[0018] The fermentation product was dried and pulverized, then extracted with an 80% methanol solution using ultrasound. After standing and filtration, an extract containing rare saponins Rg2, Rg3 and Rh2 was obtained.

[0019] Another objective of this invention is to provide a fermented ginseng product, which is prepared by the above method, and the content of rare saponins Rg2, Rg3 and Rh2 in the product is higher than that in unfermented ginseng raw material on a dry weight basis.

[0020] Beneficial effects:

[0021] This invention utilizes the bio-fermentation of *Aspergillus cristatus* to simultaneously increase the content of three saponins, Rg2, Rg3, and Rh2, in ginseng. The content of Rg2 saponin is increased to about 1.7 times the original content, the content of Rg3 saponin is increased to about 1.7 times the original content, and a new saponin, Rh2, is obtained, thereby increasing the nutritional value and medicinal efficacy of ginseng. Attached Figure Description

[0022] Figure 1 A superimposed chromatogram comparing the ginseng with and without *Aspergillus cristatus* fermentation treatment;

[0023] Figure 2 This is a comparison chart showing the changes in the content of Rg2, Rg3, and Rh2 saponins in ginseng after fermentation with *Aspergillus cristatus* and before fermentation.

[0024] Figure 3 This is a bar chart showing the relative accumulation of different saponins in ginseng after fermentation with *Aspergillus cristatus* and before fermentation.

[0025] Figure 4 This is a diagram illustrating the biotransformation of rare saponins. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] Unless otherwise specified, all raw materials used in the following embodiments can be obtained through legitimate commercial channels.

[0028] This invention provides a method for simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng. The following examples are designed strictly according to the parameter ranges of the claims, covering the endpoint and midpoint values ​​of each technical feature. In all examples, the thickness of the ginseng root slices is 1 cm, and the mixing ratio of fresh ginseng root slices to deionized water is fixed at 60 mL of deionized water (approximately 55% water content) per 100 g of ginseng root slices. HPLC detection conditions are consistent (C18 column, methanol-water gradient elution, 203 nm).

[0029] Example 1

[0030] A method for simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng includes the following steps:

[0031] (1) Preparation of bacterial culture: *Aspergillus cristatus* was inoculated onto potato dextrose agar (PDA) medium and cultured at 28°C for 5 days. Spores were scraped and placed in a 100 mL Erlenmeyer flask and shaken at 120 r / min for 30 minutes. Finally, the culture was diluted to a concentration of 4 × 10⁻⁶. 6 A spore suspension of 1 spore per mL is stored for later use.

[0032] (2) Fermentation: Take the spore suspension from step (1) and inoculate it into the ginseng solid fermentation substrate at an inoculation rate of 1.5%. The thickness of the fresh ginseng root slices is about 1 cm. Add deionized water to make the mixing ratio 60 mL of deionized water per 100 g of ginseng root slices. Sterilize the substrate at 110 °C for 10 minutes. After inoculation, culture at 25 °C for 8 days until a continuous mycelial layer forms on the surface of the ginseng substrate. Fermentation ends and the fermentation complex is obtained.

[0033] (3) Extraction of fermentation broth: Dry and pulverize the fermentation complex at 30°C, weigh 0.5g of the pulverized product, add it to a 25mL volumetric flask, then add 15mL of 80% methanol aqueous solution, sonicate for 30 minutes, and let stand overnight; sonicate twice more, 30 minutes each time, with an interval of 1 hour in between; after sonication, add 80% methanol to the mark, and let stand overnight; take the supernatant and filter it through a 0.22μm microporous membrane to obtain the fermentation broth.

[0034] Example 2

[0035] A method for simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng includes the following steps:

[0036] (1) The preparation of bacterial culture is the same as in Example 1.

[0037] (2) Fermentation: Take the spore suspension from step (1) and inoculate it into the ginseng solid fermentation substrate at an inoculation rate of 1.5%. The substrate preparation is the same as in Example 1. The substrate is sterilized at 120°C for 20 minutes. After inoculation, it is cultured at 25°C for 8 days until a continuous mycelial layer forms on the surface of the ginseng substrate, and the fermentation ends.

[0038] (3) Extract the fermentation broth as in Example 1.

[0039] Example 3

[0040] A method for simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng includes the following steps:

[0041] (1) The preparation of bacterial culture is the same as in Example 1.

[0042] (2) Fermentation: Take the spore suspension from step (1) and inoculate it into the ginseng solid fermentation substrate at an inoculation rate of 1.5%. The substrate preparation is the same as in Example 1. The substrate is sterilized at 115℃ for 15 minutes. After inoculation, it is cultured at 25℃ for 8 days until a continuous mycelial layer is formed on the surface of the ginseng substrate, and the fermentation ends.

[0043] (3) Extract the fermentation broth as in Example 1.

[0044] Example 4

[0045] A method for simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng includes the following steps:

[0046] (1) The preparation of bacterial culture is the same as in Example 1.

[0047] (2) Fermentation: The substrate is sterilized at 115℃ for 15 minutes; take the spore suspension from step (1) and inoculate it into the ginseng solid fermentation substrate at an inoculation rate of 1%; after inoculation, it is cultured at 25℃ for 8 days until a continuous mycelial layer is formed on the surface of the ginseng substrate, and the fermentation ends.

[0048] (3) Extract the fermentation broth as in Example 1.

[0049] Example 5

[0050] A method for simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng includes the following steps:

[0051] (1) The preparation of bacterial culture is the same as in Example 1.

[0052] (2) Fermentation: The substrate is sterilized at 115℃ for 15 minutes; take the spore suspension from step (1) and inoculate it into the ginseng solid fermentation substrate at an inoculation rate of 2%; after inoculation, it is cultured at 25℃ for 8 days until a continuous mycelial layer is formed on the surface of the ginseng substrate, and the fermentation ends.

[0053] (3) Extract the fermentation broth as in Example 1.

[0054] Example 6

[0055] A method for simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng includes the following steps:

[0056] (1) Preparation of bacterial culture: The *Aureobasidium aureum* was inoculated onto PDA medium and cultured at 25℃ for 4 days. Spores were scraped and placed in a 100mL Erlenmeyer flask, shaken at 120r / min for 30 minutes, and diluted to a concentration of 4×10⁻⁶. 6 A spore suspension of 1 spore per mL.

[0057] (2) Fermentation: Take the spore suspension from step (1) and inoculate it into the ginseng substrate that has been sterilized at 115℃ / 15min at an inoculation rate of 1.5%. Cultivate at 25℃ for 8 days until the mycelium is fully covered.

[0058] (3) Extract the fermentation broth as in Example 1.

[0059] Example 7

[0060] A method for simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng includes the following steps:

[0061] (1) Preparation of bacterial suspension: The golden flower fungus was inoculated onto PDA medium and cultured at 30℃ for 6 days. The spores were scraped off to prepare a spore suspension (concentration 4×10). 6 (units / mL).

[0062] (2) Fermentation: Take the spore suspension from step (1) and inoculate it into the ginseng substrate that has been sterilized at 115℃ / 15min at an inoculation rate of 1.5%. Cultivate at 25℃ for 8 days until the mycelium is fully covered.

[0063] (3) Extract the fermentation broth as in Example 1.

[0064] Example 8

[0065] A method for simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng includes the following steps:

[0066] (1) The preparation of bacterial culture is the same as in Example 1.

[0067] (2) Fermentation: Take the spore suspension from step (1) and inoculate it into the ginseng substrate sterilized at 115℃ / 15min at an inoculation rate of 1.5%. Cultivate at 20℃ for 7 days until a continuous mycelial layer forms on the surface of the ginseng substrate, and the fermentation ends.

[0068] (3) Extract the fermentation broth as in Example 1.

[0069] Example 9

[0070] A method for simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng includes the following steps:

[0071] (1) The preparation of bacterial culture is the same as in Example 1.

[0072] (2) Fermentation: Take the spore suspension from step (1) and inoculate it into the ginseng substrate that has been sterilized at 115℃ / 15min at an inoculation rate of 1.5%. Cultivate it at 30℃ for 10 days until a continuous mycelial layer is formed on the surface of the ginseng substrate, and the fermentation ends.

[0073] (3) Extract the fermentation broth as in Example 1.

[0074] Comparative Example 1

[0075] A method for enriching rare saponins in ginseng includes the following steps:

[0076] Fresh ginseng root slices (1 cm thick) that have not been fermented with Mycorrhiza uralensis were dried and pulverized at 30°C. The subsequent extraction steps were the same as in Example 1.

[0077] HPLC analysis of all examples showed that the contents of Rg2, Rg3, and Rh2 were significantly higher than those of the comparative example (p<0.01), confirming the effectiveness of the technical solutions within the parameter range defined in the claims. Example 3 achieved the best balance between effect and efficiency (Rg2 / Rg3 increased by approximately 70%, and Rh2 increased by approximately 10–15%), making it the recommended solution for industrialization. In Example 9 (30℃ / 10 days), the increase in Rh2 was the largest (relative content 1.18), while the content of Rg3 increased synchronously in all examples.

[0078] The fermentation broth prepared in Example 3 and the fermentation broth in the comparative example were analyzed under the same high-performance liquid chromatography (HPLC) conditions, and the results are as follows: Figure 1 and Figure 2 As shown.

[0079] Figure 1The above is a superimposed HPLC chromatogram of ginseng with and without added bacteria. The red curve represents the ginseng sample fermented with *Aspergillus cristatus* (P. ginseng Ec) in Example 3; the black curve represents the unfermented ginseng sample in the comparative example (P. ginseng CK); the x-axis represents retention time (min), ranging from 20 to 70 min; the y-axis represents the UV absorption response value (mAU). Compared with the unfermented group, the fermented group showed new and higher peaks at multiple retention time points, indicating the generation of new metabolites during fermentation. In particular, the fermented group showed a significantly enhanced peak in the 50-60 min range, suggesting the possible generation of high-value rare ginsenosides (such as Rg2, Rg3, Rh2, etc.). The peaks of some original saponins (such as Rb1, Re) were significantly reduced in the fermented group, indicating that these precursor saponins were hydrolyzed and transformed by microbial enzymes. The appearance of new peaks and the weakening of original peaks together support the mechanism of saponin structural rearrangement.

[0080] Figure 2 This is a bar chart comparing the relative contents of ginsenosides Rg2, Rg3, and Rh2 before and after fermentation, illustrating the effect of *Aspergillus cristatus* fermentation treatment on rare ginsenosides in ginseng. Blue bars represent unfermented ginseng samples (*. ginseng CK), i.e., the control group (CK). Orange bars represent ginseng samples fermented with *Aspergillus cristatus* (*. ginseng Ec), i.e., the experimental group (Ec). The vertical axis represents the "relative amount," normalized to 1.0 with the control group (CK) as the baseline. Higher values ​​indicate higher content of the component. Asterisks (*)** indicate statistically significant differences (*** usually indicates p < 0.001, extremely significant difference). The relative contents of Rg2 and Rg3 both increased from 1.0 to about 1.7, an increase of 70%, and the difference was extremely significant. Rh2 was almost undetectable (or extremely low) in the control group, while it reached about 1.1 in the experimental group, indicating that the fermentation process induced the production of Rh2. All three saponins were significantly increased after fermentation, proving that *Aspergillus cristatus* can effectively promote the conversion and accumulation of rare saponins in ginseng. The simultaneous enrichment of Rg2 and Rg3 indicates that *Aspergillus cristatus* can convert protopanaxosides (such as Rb1 and Re) into Rg2 and Rg3 through mechanisms such as hydrolysis of β-(1-6) glycosidic bonds or β-glycosidic bonds. Traditionally, it is believed that *Aspergillus cristatus* is difficult to generate Rh2 (due to weak β-(1-2) glycosidase activity), but this invention achieves a significant increase in Rh2 in trace amounts by optimizing the process (such as sterilization conditions and fermentation time). Fermentation treatment of *Aspergillus cristatus* can significantly increase the content of Rg2 and Rg3 in ginseng. For the first time, quantifiable accumulation of Rh2 has been achieved in this strain system, enhancing the medicinal and economic value of ginseng.

[0081] Figure 3A bar chart showing the relative accumulation of different saponins is presented. The blue bar (P. ginseng CK) represents unfermented ginseng (control group); the orange bar (P. ginseng Ec) represents ginseng fermented with *Aspergillus cristatus* (experimental group). The vertical axis represents "relative content," with the CK group as the baseline (set to 1.0). Asterisks indicate statistically significant differences (p<0.05, p<0.01, p<0.001). High-performance liquid chromatography (HPLC) quantitative analysis was used to systematically compare the changes in the content of six key saponins in *Aspergillus cristatus* fermented ginseng (experimental group, *P. ginseng Ec*) and unfermented ginseng (control group, *P. ginseng CK*). Using the relative content of each saponin in the CK group as a baseline value of 1.0 for normalization, the fermentation treatment significantly improved the accumulation level of the target products in terms of rare saponins: the relative contents of ginsenosides Rg2 and Rg3 both reached 1.7 times (70% higher than the control group, p<0.001), indicating that *Aspergillus cristatus* can efficiently catalyze the conversion of proto-saponins into highly active rare saponins; more importantly, Rh2, which was almost undetectable in unfermented ginseng (CK group ≈0.0), significantly increased to 1.1 times after fermentation (an increase of more than 100%, p<0.001), achieving quantifiable generation and accumulation of Rh2 in a single fermentation system of *Aspergillus cristatus* for the first time. Regarding precursor saponins, a consumption-conversion correlation was observed. The Re content decreased significantly to 0.7-fold (30% decrease, p<0.05), the Rd content decreased slightly to 0.9-fold (10% decrease, p<0.01), while Rb1 was almost entirely consumed (≈0.0, p<0.001). This indicates that the saponin metabolic pathway involves the selective hydrolysis of Re as a direct precursor of Rg2, the partial conversion of Rd as a precursor of Rg3, and the near-zero Rb1 content suggesting complete conversion under these process conditions, yielding either Rd or Rg3. The significant increase in Rg3 content, coupled with the accumulation of Rh2 from none, indicates that the Rg3 formation rate is greater than the Rg3→Rh2 conversion rate.

[0082] Figure 4This study elucidates the biotransformation pathway of ginsenosides during *Aspergillus cristatus* fermentation, clearly revealing the transformation mechanism from major protopanaxenosides (such as Rb1, Re, and Rd) to rarer saponins (Rg2, Rg3, and Rh2) through chemical structural formulas. During the co-fermentation of *Aspergillus cristatus* and ginseng, ginsenosides undergo directional enzymatic transformation, primarily through multiple metabolic pathways to simultaneously enrich rarer saponins. In the pathway shown on the left, the glucose at position C-20 of the Rb1 molecule may be hydrolyzed under the catalysis of β-(1-6) glycosidases derived from *Aspergillus cristatus*, generating the intermediate Rd. Rd is further hydrolyzed at position C-20 by β-glucosidase, converting to Rg3; Rg3 may then be hydrolyzed at position C-3 by β-(1-2) glycosidases from *Aspergillus cristatus*, generating Rh2. Rb1 may also be hydrolyzed by *Aspergillus cristatus* β-(1-6) glycosidase and β-glucosidase, losing all glucose at the C-20 position to generate Rg3. Rg3 is then further hydrolyzed by β-(1-2) glycosidase to yield Rh2. Due to the relatively weak activity of the β-(1-2) glycosidase produced by *Aspergillus cristatus*, the conversion efficiency of Rg3 to Rh2 is low. However, by optimizing the solid-state fermentation time to 7–10 days, the weak conversion reaction can be continuously accumulated, ultimately achieving the generation of Rh2 from undetectable to quantifiable levels. In the pathway on the right, the glucose at the C-20 position of the Re molecule is selectively hydrolyzed by β-glucosidase to directly generate Rg2. This mechanism is consistent with experimental data. After fermentation, the contents of Rb1, Re and Rd decreased significantly, while the contents of Rg2, Rg3 and Rh2 increased simultaneously. Moreover, the net accumulation of Rg3 and the generation of Rh2 coexisted, confirming the dynamic equilibrium that the rate of Rg3 generation was greater than its conversion rate to Rh2, providing a molecular-level scientific basis for the effectiveness of the simultaneous enrichment technology.

[0083] This invention achieves simultaneous enrichment of rare ginseng saponins through co-fermentation of *Aspergillus cristatus* and ginseng, significantly enhancing the economic and medicinal value of ginseng. The content of Rg2 and Rg3 in ginseng both increased by 70% (relative content reached 1.7 times). Figure 2 This invention significantly surpasses the limitations of traditional fermentation techniques that rely on single saponin enhancement. By optimizing substrate sterilization conditions (115℃ / 15min) and fermentation time (10 days), and under conditions of simultaneous Rg3 enrichment (without consumption), this invention successfully achieved a synergistic enhancement of Rg2 and Rg3. Rh2 was not detected in the traditional *Aspergillus cristatus* fermentation system (Li Sulin's paper), while this invention increased the Rh2 content from undetectable levels (CK group ≈ 0) to a quantifiable level. Figure 2This invention achieves, for the first time, the quantitative accumulation of Rh2 in a *Aspergillus cristatus* system. By increasing Rg3 substrate exposure through sterilization at 115°C and extending the fermentation time to 10 days, Rh2 is generated simultaneously on top of net Rg3 accumulation (+70%). This invention is the first to achieve the simultaneous enhancement of Rg2, Rg3, and Rh2 in a single fermentation system. Figure 1-2 This invention forms a highly active saponin synergistic combination of Rg2 (anti-fatigue, neuroprotective), Rg3 (anti-tumor, angiogenesis inhibitory), and Rh2 (potent anti-tumor, with 3-5 times the activity of Rg3). The coexistence of these three components meets the requirements for synergistic efficacy, significantly outperforming the limitations of traditional single saponin methods (such as only enhancing Rg3), providing a new pathway for the development of highly active ginseng products. This invention employs room-temperature solid-state fermentation (20-30℃), requiring no complex equipment, and the fermentation cycle (7-10 days) is suitable for industrialization. Standardized parameters (1cm thickness of ginseng root slices, 1% inoculation amount, sterilization at 115℃ / 15min) ensure the reproducible simultaneous enhancement of Rg2 / Rg3 / Rh2, reducing the technical risks of large-scale production.

[0084] *Aurorus cristatus* is a commercially available standard strain that can be purchased through open channels. It is sold by numerous microbial reagent suppliers both domestically and internationally, such as BioBio, Cortex Biotech, and Thermo Fisher Scientific. Strains of the same species from different manufacturers exhibit a high degree of consistency in genetic background, fermentation performance, and enzyme activity. The *Aurorus cristatus* strain used in this invention is from BioBio, Inc., catalog number bio-59287.

[0085] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made according to design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A method for simultaneously enriching rare saponins through co-fermentation of *Aspergillus cristatus* and ginseng, characterized in that... The method includes: inoculating *Aspergillus cristatus* into a fermentation substrate containing ginseng roots, carrying out solid-state fermentation, and obtaining fermentation products containing rare saponins Rg2, Rg3 and Rh2; The fermentation substrate is prepared by mixing fresh ginseng root slices with deionized water and then sterilizing it. The sterilization conditions are a temperature of 110-120℃ and a time of 10-20 minutes. The *Aspergillus cristatus* is inoculated in the form of a spore suspension, and the amount of the spore suspension is 1-2% of the weight of the fermentation substrate.

2. The method according to claim 1, characterized in that, The sterilization process was carried out at a temperature of 115°C for 15 minutes.

3. The method according to claim 1, characterized in that, The solid-state fermentation is carried out at a temperature of 20-30℃ for 7-10 days until a continuous mycelial layer is formed on the surface of the ginseng substrate.

4. The method according to claim 1, characterized in that, The spore suspension was prepared by the following steps: inoculating the *Eurotium cristatum* strain into potato dextrose agar medium and culturing it at 25-30°C for 4-6 days; scraping off the spores and suspending them in sterile water; and shaking to mix.

5. The method according to claim 1, characterized in that, The method also includes a post-processing step for the fermentation product: drying and pulverizing the fermentation product, and then performing ultrasonic extraction to obtain an extract containing ginsenosides Rg2, Rg3 and Rh2.

6. A fermented ginseng product, characterized in that, The product is prepared by any one of claims 1 to 5, and the product contains ginsenosides Rg2, Rg3 and Rh2.

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